1. Field of the Invention
The present invention relates to an apparatus and method for differentiating erythrocytes in urine, and more particularly to an apparatus and method using flow cytometry for differentiating erythrocytes in urine to determine the origin and type of the erythrocytes.
2. Description of the Related Art
There are two cases that give rise to hematuria, where erythrocytes appear in urine. Hematuria of glomerular origin is caused by an internal disease such as nephritis, whereas hematuria of non-glomerular origin occurs by a urinary disease such as bladder cancer, kidney cancer, and urethral calculi. When hematuria is observed, it is necessary to search for the cause thereof.
Methods conventionally known for determining whether erythrocytes in urine are of glomerular origin (an internal disease) or non-glomerular origin (a urinary disease) are:
(a) using a microscope to differentiate erythrocytes in accordance with their morphological differences, and
(b) using an automatic blood cell counting apparatus to differentiate erythrocytes in accordance with the differences in their size.
However, according to method (a), there is a problem that inspection for morphological differences is complicated and takes a lot of time. Also, it is difficult to maintain reliability of the inspection because it is based on human eye inspection which requires expertise.
According to method (b), there is a problem that a pretreatment such as centrifugal separation is required because it is necessary to make measurements with the electric conductivity of a urine specimen maintained at a constant value. Also, there is a fear that the accuracy of judgment decreases by mistakenly identifying what is not an erythrocyte (for example, a bacterium or a crystalline component) as an erythrocyte.
In order to solve the above-mentioned problem, there is known (c) a new apparatus which differentiates erythrocytes, leukocytes, epithelial cells, urinary casts, and bacteria by processing the optical signal intensities of scattered light and fluorescent light of the particles as obtained from a flow cytometer, and analyzing erythrocytes in urine in accordance with a bias state of a particle size distribution of the differentiated erythrocytes (See Japanese Unexamined Patent Publication No. Hei 8(1996)-240520).
This apparatus recognizes that the erythrocytes of glomerular origin generally have smaller particle sizes than those of non-glomerular origin, whereby the group of erythrocytes whose particle size distribution is biased to a smaller size is classified as glomerular-origin erythrocytes, and the group of erythrocytes whose particle size distribution is biased to a larger size is classified as non-glomerular-origin erythrocytes.
Specifically, a calculating section of the apparatus calculates a number Raz of erythrocytes falling within a range from La to Lz, a number Ra2 of erythrocytes falling within a range from La to L2, and a number R1z of erythrocytes falling within a range from L1 to Lz, provided that La represents a lower limit of particle sizes in the erythrocyte size distribution, Lz represents an upper limit thereof, and L1 and L2 represent preset values satisfying the relation La&lt;L1&lt;L2&lt;Lz.
A determining section of the apparatus determines the erythrocytes to be of glomerular origin when Ra2/Raz is greater than a first predetermined value and determines the erythrocytes to be of non-glomerular origin when R1z/Raz is greater than a second predetermined value. If neither of the above inequality relationships are satisfied, the erythrocytes are determined to be of mixed kind. If both of the above inequality relationships are satisfied, the erythrocytes are determined to be of non-glomerular origin.
The judgment in the prior art method (c) has been devised by considering that, although the particle size distribution of erythrocytes of non-glomerular origin has a peak biased to a larger size and the particle size distribution of erythrocytes of glomerular origin has a peak biased to a smaller size, there is an overlapping region (region where the two particle size distributions overlap with each other). In other words, the erythrocytes of non-glomerular origin are captured by using the ratio of erythrocytes having a particle size larger than a preset value including the overlapping portion, and the erythrocytes of glomerular origin are captured by using the ratio of erythrocytes having a particle size smaller than a preset value including the overlapping portion, so as to determine the origin of the erythrocytes.
However, the judgment actually carried out in an inspection room often did not coincide with the observation result obtained by microscopy.
This may have been brought about by the following reason. In this judgment, the erythrocytes that have a small size are determined as erythrocytes of glomerular origin, assuming that the erythrocytes of non-glomerular origin have a morphology similar to those in blood. However, the erythrocytes may be damaged in urine to give a signal that indicates a smaller size. The term "damage" as used herein refers to a damage of erythrocytes occurring in aciduria, hyposthenuria, or the like while the erythrocytes are in a bladder or in a urine specimen after urination.
Here, the term "glomerular origin" refers to urine containing a large proportion of deformed erythrocytes. The deformed erythrocytes are red blood cells that are deformed to have a characteristic shape by passing through a glomerulus of a kidney, and have a morphology different from that of damaged erythrocytes appearing in aciduria, hyposthenuria, or the like. In other words, the erythrocytes can be differentiated by the prior art method only under a condition in which the erythrocytes have not been damaged in urine.